paper no. : 09 animal cell biotechnology module :10

Biotechnology

Animal Cell biotechnology

Transgenic animal and methods of their creation

Paper No. : 09 Animal Cell Biotechnology

Module :10 Transgenic animal & method of their creation

Principal Investigator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director

The Energy and Resources Institute (TERI), New Delhi

Paper Coordinator: Dr. Minakshi, Professor & Head, Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar

Content Writer: Dr. Anil Kumar, Assistant Professor, CMBT Maharshi Dayanand University, Rohtak

Paper Reviewer: Dr. Rashmi Bhardwaj, CMBT, Maharshi Dayanand

University, Rohtak

Co-Principal Investigator: Prof S K Jain, Professor, of Medical Biochemistry

JamiaHamdard University, New Delhi

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Description of Module

Subject Name Biotechnology

Paper Name Animal Cell Biotechnology

Module Name/Title Transgenic animal and methods of their creation

Module Id 10

Pre-requisites Basic knowledge of genetic engineering and molecular biology

Objectives

1. To Know about the basic concept of transgenic animals.

2. To Learn about method of transgenic animal creation.

3. To Learn about ethical, social and legal concern related to transgenic

animals.

4. To Know about applications of transgenic animals in medicine, agriculture

and industrials.

Keywords Genetic engineering; Transgenic technology; Transgenic animal; Transgene

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Contents

1.0 Introduction

2.0 History

3.0 Methods

3.1 Construction of transgene

3.2 Gene/transgene transfer methods

3.2.1 DNA microinjection

3.2.2 Embryonic stem cell mediated gene transfer

3.2.3 Nuclear cloning transfer

3.2.4 Retroviral mediated gene transfer

3.2.5 Sperm mediated gene transfer

4.0 Examples of transgenic technology

4.1 Transgenic mice

4.2 Transgenic livestock

4.3 Transgenic pigs

4.4 Transgenic fish

5.0 Applications of transgenic technology

5.1 Medical applications

5.2 Agricultural applications

5.3 Industrial applications

6.0 Ethical concerns

7.0 Summary

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1.0 INTRODUCTION

In the mid 1980s, onset advancements in molecular genetics had changed mankind’s ability to

manipulate biological organisms at genetic level, physiological level and cell biological level. These

techniques collectively are referred as Biotechnology, the newest yet the most controversial field in

science technology. Biotechnology came into existence in 1970s after the emergence of genetic

engineering, the manipulation at genetic level to produce modified organisms including plants and

animals. The mutual contributions of these two approaches have given thescientists the opportunity to

modify animals for increasing the efficiency of their production. It can be done by isolating and

transferring gene/genes from one organism to another organism for the expression of desirable trait.

This modification led to the development of most complex aspect in the field of genetic engineering,

known as“transgenic technology”.

“Transgenic technology”, also known as transgenesis, is a process of integration of exogenous genes

or cloned DNA into genetic material of an organism producing DNA alterations to give permanent

change of effect. Organisms containing integrated sequences of DNA are called “Transgenic Animal”

or “Genetically Modified Organisms (GMO)”. Foreign genes which insert itself into the germ line of

the animal and transmitted to the progeny are called as “transgenes”. Transgenesis may involve in

vivo alteration of body function of an organism, rather than individual cells. So, the ability of

transgenesis to introduce genes into eggs or early embryos has given an extension to embryological

study providing scope for the production of farm animals of desirable traits. Therefore, transgenesis

had been proved to be far better and complex than cell-culture based applications by imparting

knowledge at embryological level.

Transgenic animals are used for variety of purposes such as in research areas, to produce valuable

drugs for animals/human welfare, to generate animal models and cell lines for studying pathogenesis

and/or prevention of disease in humans, to study genetic regulation of development and growth, to

produce conventional products in more quantity (e.g. meat, milk, leather, wool etc.) and for the

synthesis of non-conventional products e.g Interferon, ᾳ- antitrypsin etc.

This sector of genetic engineering has raised a great public concern, equal in both consequences and

potential including the outlined basic research technologies toward fruitful applications. The

development and use of transgenic organisms involve many complex surrounding issues. In addition,

in higher organisms many scientific and technical problems are associated with genetic engineering

that is often difficult to handle. The problem rise due to genome complexity and difference in

development pattern of plants and animal. Despite of all these hurdles, methods for transgenics are

now well established. In this module, considering basic concept of transgenic animals, we will learn

how to produce transgenic animals, their applications in medicine, agriculture and industrials fields

and their ethical, social and legal concerns.

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2.0 HISTORY

The first genetically modified organism was a bacterium, Escherichia coli. Since then, technique of

genetic manipulation of organisms had a remarkable progress in production of GMOs in lower as well

higher organisms. Three decades ago Jaenisch and collaborators produce genetically modified mouse

that represent one of milestone in genetic engineering. Even today, the mouse is the most preferable

model organism for genetic studies. The mouse genome sequencing was completed in 2002 served as

base for genetic manipulations in livestock animals, such as cattle, pig, sheep andgoats (Table 1).

Table 1: Historical achievements in transgenesis

YEAR IMPORTANT LANDMARKS IN TRANSGENIC ANIMALS

1985 Transgenic pig

1986 Embryonic cloning in sheep by nuclear transfer

1991 Transgenic dairy cattle

1992 Transgenic pigs with enhanced resistance to viral infection

1994 Pig with ability to express inhibitor of human complement system

1997 Dolly (sheep) was produced using somatic cell nuclear transfer techinque

Production of transgenic livestock as a model for human disease

1998 Production of transgenic cattle by use of nuclear transfer technology

2000 Gene targeting was used for transgenic sheep production

2001 “ecologically correct” transgenic pig

2002 Biopolymer fiber production from transgenic animals

Transgenic calf with ability to produce human artificial chromosome

2003 Transgenic cattle with altered milk proteins component

2004 Inactivation of two bovine genes by sequential method

2005 Production of transgenic cow with resistance to bacterial infection

3.0 METHODS

Several transgenic approaches have been developed for the creation of transgenic animals, each of

which has its own advantages and disadvantages. The development of transgenic animals is mainly

divided into two parts –

1) Construction of Transgene,

2) Introduction of transgene into animal.

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VECTO

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Paper Reviewer: Dr. Rashmi Bhardwaj,

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Further, introduction of transgene into animals is to be done. This intentional introduction

of gene into a living organism can be done via five principal methods known as “gene

transfer methods” which are –

a) DNA microinjection

b) embryonic stem-cell mediated transfer

c) Nuclear cloning transfer

d) Gene transfer using retrovirus and sperm

These techniques provide better opportunities for development of transgenic

animals for breeding purposes and their subsequently use in medical sciences and livestock

production.

3.1 CONSTRUCTION OF A TRANSGENE

A ‘transgene’ is like any other gene which indirectly codes for a protein corresponding

to a particular trait. Because of the universality of genetic code, a transgene inserted

into desirable transgenic organism will allow to produce the same protein (and therefore

exhibit the same trait) as the original, “donor” organism.Transgenes are introduced into

the recipient organism’s germ line, so that it is transferred to organism’s progeny. In

order to transfer transgene from one organism to other organism, it needs to be

constructed manually. Using recombinant DNA methods, transgenic DNA can

be created with predictable expression in the animal. Transgene contain 3 parts –

o Promoter

o Gene to be expressed

o Termination sequence

Use of restriction enzymes and ligase allows us to combine functional regions from

different species in a test tube. Vector DNA allow integration of transgene into

host DNA and regulatory sequences (promoter and enhancer) ensure proper functioning

of the gene within the host genome.

All components of endogenous DNA can be isolated and recombined to form a

transgene expression cassette or construct (Fig. 1).

Fig. 1. Linear transgene DNA construct. A cDNA of gene of interest is used. The

promoter and 3’ unstranslated region must be included for proper regulation. Somehow,

intron also plays role in gene regulation, so a portion of it is included.

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3.2 GENE/TRANSGENE TRANSFER METHODS

In higher plants, or even, in bacteria and other microbes the uptake of genes by cells is often described

as 'transformation'. While in animals the term is replaced by the term ‘gene transfer’

or 'transfection' because the term transformation is used in animals to describe phenotypic alteration in

cells. Therefore, Transfection, also called gene transfer methods, is a procedure that introduces

foreign genes (transgenes) into living organism to produce genetically modified organisms or

transgenic animals.

In last few decades, a wide repertoire of gene transfer techniques has been evolved in field of

transgensis. One of which is physical transfection. It enables the direct transfer of nucleic acids

(DNA/RNA) by physical or mechanical means into the cytoplasm, or nucleus without the usage of

foreign substances like lipids. It is most preferable method for gene transfer as it avoids the

complications associated with viral and chemical strategies. Physical transfection is of further 3 types:

3.2.1 DNA MICROINJECTION

DNA microinjection was first introduced in early 1900 by Dr. Marshall A. Barber. They showed that

a foreign DNA can be incorporated and expressed into single-cell pronuclei embryos, and can be

transmitted to the offspring of transgenic mice. This is most widely accepted method for generation of

transgenic animals, particularly mammals. It involves manipulation of single living cell by directly

injecting naked DNA, mRNA or proteins from another member of same or different species through a

fine glass of micropipette. This process of microinjection is generally carried out in pronucleus of

fertilized ovum, but can also be done in egg, oocyte, embryonic stem cell or embryos of animals. The

transgene may cause over or under expression of certain genes.

The eggs are removed from superovulated female. Male and female pronucleus is allowed to fertilize

in vitro to produce fertilized ovum. The transgene delivery is done using a glass micropipette with a

fine tip of 0.5 mm (or µm) in diameter under a powerful microscope. During microinjection, cells of

interest to be microinjected into the cell nucleus or cytoplasm of adherent cells (fertilized ovum) are

placed in a vial. A holding pipette under the view of the microscope sucks and holds the target cell at

the tip which is injected into cell membrane to deliver the contents of the needle into the cytoplasm.

The modified fertilized ovum is, then, implanted into the oviduct of foster mother (recipient female).

The technique of microinjection has been shown in Fig. 2.

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Fig. 2: DNA microinjection [adapted from molecular biotechnology: principles and applications of

recombinant DNA by Glick et al., 2010]

Efficiency rate of this method is probably very low (1 to 30 %), only 1 out of 3 implanted embryos

survive till birth. We can confirm animal is carrying a gene of interest or not by analyzing tissue

sample taken from tail after their birth. Less than 20% of offspring will be produced in heterozygous

condition (present in only one copy of the gene) and mating two heterozygous animals result in 25%

percent of homozygous offspring having two copies of desired gene from both parents.

DNA microinjection does provide stable integration after several hundred copies of DNA segment are

utilized, therefore, it is labor-intensive. So, it requires skills to give reproducible results on routine

basis. Moreover, there is high probability that the injected gene may not insert itself into host DNA at

desired site.. Microinjection is technically time consuming and expensive. It is applicable for wide

range of species, but most commonly used for production of transgenic mice.

3.2.2 EMBRYONIC STEM-CELL MEDIATED TRANSFER

The term embryonic stem cell (ESC) was first used in 1981 to denote a cell line isolated directly from

mouse embryo. ESC are harvested from inner cell mass (ICM) of early mouse embryos from blastula

stage. They have the potential to participate in embryogenesis with high efficiency when combined

with normal embryos to form chimaeras. Their efficiency is maintained even after they are altered or

manipulated for gene transfer and can retain their full potential when grown in culture to differentiate

into all cells type. Due to this reason, it provides an excellent vehicle for gene transfer into living

organism. This is known as ESC mediated gene transfer (Fig.3).

This method is most common in mice. Initially, blastocysts are isolated from blastula stage from

mouse embryo. Inner cell mass (ICM) is removed from blastocysts and cultured with embryonic stem

cDNA

promoter

any

UTR

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cells. DNA molecules (transgene) containing desired structural gene is constructed by using

recombinant DNA method with promoter and enhancer sequences.

Fig. 3: Embryonic stem cell mediated transfer [adapted from molecular biotechnology: principles and

applications of recombinant DNA by Glick et al., 2010]

ES cells are transformed by exposing cultured cells to the foreign DNA. Like pronuclear

microinjection, introduction of DNA into ES cells is also random. If the injected DNA is having

similar sequence to part of the mouse genome, it may undergo "homologous recombination" and

integrate itself at a specific site as a single copy. Successfully transformed ES cells are then selected

and injected into cavity of host blastocysts of different mouse strain and re-implanted into uterus of

foster mother (pseudo-pregnant mother).

After the birth of offspring, they are tested for desirable gene by examining their small part of tissue

obtained from tail. An ES cell colonizes host embryo and contributes to the germ line. If ES cells have

contributed to their germ cells, atleast 10 – 20% will show positive results with heterozygous

condition in F1 generation. To establish strain, homozygous condition is produced by mating two

heterozygous offsprings (F2 generation). Mating of heterozygous produces transgenic animal..

Embryonic stem-cell mediated gene transfer is time and cost intensive method but holds great

importance. It helps in studying genetic control during developmental process by allowing the

detection of transgenesis at embryonic level. Due to this, mouse genome can also be modified at

3

’

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generation

0.5mm

diameter

micropipet

(from

vasectomized

male)

Transge

nic

mice is,

then,

bred to

Identifica

tion by

analyzing

tail of the 1

2

3

4

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embryonic level by inserting, removing or modifying DNA sequences, which could lead to Knock-

out, knock-in or conditional mutant mice.

3.2.3 NUCLEAR CLONING TRANSFER

Nuclear cloning transfer, also known as somatic cell nuclear transfer (SCNT), is one of gene transfer

method established to overcome the shortcomings of other transgenic techniques. This technique

involves transfer of somatic cell nucleus (i.e. Donor) to the cytoplasm of enucleated metaphase II-

oocyte (i.e. Recipient) to create a genetically identical copy, or a clone, of the somatic cell donor. This

process ensures that all the cells subjected for transformation will contain transgene and confirms 100

% produced animal are transgenic. It also provides site – specific gene expression by homologous

recombination (example –Dolly was the first female sheep created by somatic cell nucleus transfer

(SCNT) derived from differentiated mammary adult cell line (Born 5 July 1996) and other gene

targeted sheep and pigs are also produced by SCNT technique). The sex of the transgenic animal can

also be pre-determined on the basis of donor sex tissue (example – increase in the efficiency of milk

production by modification in the mammary glands of transgenic animal). The somatic cloning

process involves following steps (Fig. 4).

Somatic cells (cells other than germ cells) are collected from donor and subjected in in vitro.

Simultaneously, a mature oocyte is enucleated (recipient).

Nucleus of cultured donor cells is, then, removed and inserted in to the enucleated oocyte. The

somatic cell and oocyte are then allowed to fuse.

Fusion involves reprogramming of inserted nucleus by the host cell with the help of short high

voltage pulses through the point of contact between the two cells.

Fig. 4: Cloning by nuclear transfer in sheep

o The egg containing somatic cell nucleus, is prompted with a shock that induce its division.

5

(Transgene

(Foster

Identification by analyzing

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o Egg with somatic cell nucleus grows into blastocyst (embryo with approx 100 cells) after

many cell divisions.

o Blastocyst then implanted into foster mother, following by gestation period of nine months, a

cloned animal is born.

SCNT has the potential to generate multiple copies of transgene in a single experiment. These

transgene produces transgenic animals which can be used for pharmaceutical protein production or

xeno-transplantation. It retains genetic code of the donor nucleus that could be helpful to preserve

endangered species. Despite all advantages, overall efficiency of SCNT is very low due to high

abortion and fetal mortality rates. Its average is between 1 – 3 % in all animals including cattle. It is

because most of the embryos are lost between 35 – 60 days of pregnancy as compared to embryos

produced via in vitro fertilization. These losses are due to complications arising due to increased birth

weight, respiratory problems, developmental abnormalities and other metabolic deficiencies, which

was first observed and reported in blastomere derived nuclear transfer by Willadsen and coworkers in

1991. These complications are largely attributed to improper reprogramming of epigenetic signals

such as defects in methylation as well as gene expression as compared to in vivo and in vitro embryos.

Further efforts and new paradigms are needed to accomplish this technology to make it satisfactory

and extend it to its fullest potential.

3.2.4 RETROVIRAL MEDIATED GENE-TRANSFER

This gene transfer technique is mediated by means of a carrier (vector) such as virus or plasmid. In

this method, virus gene is replaced by transgene. It has been used in gene transfer methods due to its

infection property. Delivery of transgene to host cell is done by transfection method (gene therapy).

Retroviruses, carrying its genetic material as RNA, are most common virus or vectors used for this

purpose. The code present in retrovirus is “reverse transcribed” to produce DNA, which integrates

itself into host cell and result in chimera (Fig.5). The method allows small DNA inserts upto < 8 kb

possible and successfully reported in 1974 when a simian virus (SV) was injected into mice embryos,

resulting in chimera mice carrying transgene. The main advantage of this method is that it does not

lead to abortion and the probability of expression is much better. Transmission of virus gene is only

possible if retrovirus had integrated genes into germ cells. Expression of transgene is tested by

analyzing the animals produced in F1 generation. F1 generation usually results in chimera. Positive

results for F1 generation indicates presence of transgene in animals (heterozygous condition) which

are, then, further inbred for 10 to 20 generations in order to obtain homozygous transgenic animal. At

this phase, embryos carrying the transgene are frozen and stored till implantation.

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Fig.5: Reteroviral mediated gene transfer [adapted from molecular biotechnology: principles and

applications of recombinant DNA by Glick et al., 2010]

3.2.5 SPERM MEDIATED GENE TRANSFER (SMGT)

Lavitron et al (1989) described technique of SMGT based on the internal ability of sperm cells. Later

are referred as ‘natural vector’ of genetic material carrying exogenous DNA. This ability allows

sperm cell to bind exogenous DNA molecules in the subacrosomal region of its head, where DNA

binding proteins are already present. DNA binding proteins, then, internalises foreign DNA and

transfer them into oocyte at the time of fertilization by artificial insemination process (Fig. 6). Major

histocompatibility complex (MHC) class II and CD4 molecules are also involved in sperm/DNA

interaction. Fertilization could be in vitro or artificial insemination or waterborne fertilization (for

aquatic animals). The SMGT procedure was first introduced in small animal model, and reported high

efficiency within mouse. Later, large animals were successfully adapted and found to be highly

effective in the generation of human decay accelerating factor (HDAF) transgenic pig cell lines. It

was also successfully optimized in mutagenic transgenic pigs which contain three different reporter

genes i.e. green fluorescent protein, enhanced blue fluorescent protein and red fluorescent protein, and

was introduced.

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Fig. 6: Sperm mediated gene transfer in pigs

SMGT technique offer advantages of inexpensiveness and ease of use and high efficiency (5 – 60 %)

in contrast to 0.5 – 4 % in microinjection. It also does not require embryo handling or expensive

equipments. It provides broad applicability within species ranging from sea urchin to cattle.

4.0 EXAMPLES OF TRANSGENIC ANIMALS

4.1 TRANSGENIC MICE

Mice are the role model in transgenic technology because virtually all genes are conserved between

mouse and human. The first transgenic animal mice were created by Rudolf Jaenisch in 1974. He

inserted foreign DNA into the early- stage mouse embryos resulting in mice carrying transgene in all

their tissues. Following are different varieties of mice developed through transgenic technology are

mentioned below –

a) KNOCKOUT MICE – widely used in medical research to investigate gene function, as they

have their selected genes inactivated. They carry knock-out gene (non functional gene) at a

place of interest. These are used for genetic analysis of inherited diseases and cancer. The

transgenic mice were generated based on inheritance patterns of human disease. Therefore,

i. Introduction of collagen gene in mice genome produces knockout mice

suffering Osteogenesis. It is, then, used to treat human diseases Osteogenesis.

ii. Introduction of HIV tat gene in mice generate knockout mice with Kaposi’s

sarcoma. It is used for treatment in humans.

Since the early1980’s hundreds of different genes have been introduced into various mouse

strains. These studies have contributed to understanding of gene regulation and are also used

as human disease models for AIDS, Diabetics, Cancer, etc., diseases.

b) SUPERMOUSE – was developed by Ralph Brinster (1982). It had the ability of superior

learning and memory. It was also used for meat production. By introducing Rat growth

Transgenic founder is, then, bred to

continue in germ line

(somatic cell)

(Recipient ) (reprogramming)

(Blastocysts)

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hormone gene in supermouse, it resulted in high level of growth hormone attaining

approximately double the weight of normal offspring mouse.

c) ONCOMOUSE – It was majorly developed to study Cancer and to screen anti – tumor drugs. It

was 1st patented transgenic animal. It was made by inserting activated oncogenes for which 13

different strains were engineered to contain human oncogene causing tumor formation.

d) SMARTMOUSE – It was created to have over expression of NR@B receptor in the synaptic

pathway which increases the efficiency of mice in term of learning behaviour.

Although, development of variety of mice provided wide range of spectra in various fields but, due to

some technical problems, it also has limitations. Development of transgenic mice is quite expensive,

then development of other animals.

4.2 TRANSGENIC LIVESTOCK

Microinjection is the most common gene transfer method for transgenic livestock. It has low

efficiency as compared to transgenic mice. Examples of transgenic livestock are given below -

a) TRANSGENIC COW – ROSIE was the first transgenic cow, born in 1997. Transgenic cows

which contain two types of casein gene produce 13% more milk, which is further used for

cheese making. Also, cattles with Lactose gene insert produces milk which is poor in lactose,

which is useful for lactose intolerant people. Prion free transgenic cows aid into resistant to

mad cow disease.

b) TRANSGENIC SHEEP – Tracy (first transgenic sheep) was produced recombinant protein in

its milk. It is used for good quality of wool production. It is used as a model organism for

study human blood clotting factor IX, transplantation, manufacturing of biological products

and drug production in milk.

c) TRANSGENIC GOATS – It was mainly developed for animal farming. They are capable of

expressing tissue plasminogen activator (tPA), antithrombin III (ATryn), spider silk etc in

milk. tPA gene of plasminogen tissue also helps in dissolving blood clots. Silk genes are

transferred from spiders, in order, to produce several grams of silk protein in her milk. ATryn

is the first recombinant protein from transgenic goat that was approved in January 2009 by the

United States Food and Drug Administration (USFDA).

Development of transgenic livestock is a difficult procedure and quite expensive.

4.3 TRANSGENIC PIGS

Pigs are the only animal whose physiology matches to that of humans; therefore, transgenic pigs are

also used for Organ transplant harvesting. Transgenic pigs inserted with Human ᵦ- globulin gene,

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produces human hemoglobin in blood. It is used to treat Haemophilia. Most common example of

transgenic pig is Enviropig. Enviropigs were mainly developed to overcome digestion problems

faced by normal pigs. Normal pigs face trouble digesting phytase, found in many cereal grains of their

diet. While, transgenic pigs were produced by introduction of phytase gene obtained from E.coli to

overcome this. Benefits of this introduction is listed below –

o Phytase enzyme is present in salivary gland of transgenic pig which aids in

degradation of indigestible phytase by releasing phosphate. Pigs easily digest

phosphates.

o A genetically engineered pig has been approved for limited production which reduces

upto 65% less production of phosphorous in animal waste.

Major problem faced in the development of transgenic pig is their breeding problem including

mutations, and also releases phosphates that are harmful for environment.

4.4 TRANGENIC FISH

Transgenic fish are produced by the artificial selection and transfer of genes into fertilized eggs.

Microinjection is the desired method, though the integration rates of transgenes are generally low.

Examples are -

a) SUPERFISH, such as Salmon/trout, Tilapia, Catfish, as the name suggests were mainly

created to increase growth rate and size of normal fish. Growth hormone (GH) genes were

inserted into fertilized egg by microinjection method leading to extra copies of GH. It resulted

in increased growth and size, upto 6 times faster than control fish. Transgenic atlantic salmon

grows 10 – 11 times larger than normal fish and also been approved for consumption.

b) GLOW FISH, Zebra fish (Danio carpio), also known as ornamental GloFish. They are

produced by integrating a fluorescent protein gene derived from jelly fish into embryo of a

fish. It is the first genetically modified animal to become publicly available as a pet (contain

red, green, yellow and orange fluorescent color).

5.0 APPLICATIONS OF TRANSGENIC TECHNOLOGY

Transgenic technology has great potential in following areas

o Medical application,

o Agricultural application

o Industrial application

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5.1 MEDICAL APPLICATIONS

Transgenic animals were mainly developed as disease model for humans for studying genetic basis of

human and animal disease such as Cancer, Cystic fibrosis, Rheumatoid arthritis, Alzheimer's etc. It

also contributed in understanding mechanisms of disease resistance in humans and animals and in the

development of pharmaceutical drugs and product testing and/or screening. Transgenic cows, sheep

and goats are capable of providing nutritional supplements such as insulin, growth hormone and blood

clotting factors. Transgenic animals also provide means for organ transplantation. Patients die every

year due to lack of organs replacement (heart, liver, or kidney).Transgenic pigs can provide the

transplanted organs needed to ease the shortfall.

5.2 AGRICULTURAL APPLICATIONS

Transgenesis had made it possible to develop desired traits such as increased milk production, high

growth rate etc. by selective breeding in animals in a shorter time with more precision. It also

increases the yield production of desired characteristics. HERMAN, a transgenic bull, was developed

that carries a human Lactoferrin gene (increases iron content). Transgenic pigs and cattle weigh

approximately double than normal animals and are used for meat production. Transgenic sheep grow

heavy wool on them. Scientists are working on development of disease resistant animals such as

influenza-resistant pigs, but this phase is limited to certain genes.

5.3 INDUSTRIAL APPLICATIONS

The extraction of polymer strands from the milk helped the scientists to create light material with

toughness and flexibility that are used further for weaving military uniforms, medical micro sutures,

tennis racket strings etc. Also, wide varieties of enzymes and micro-organisms are engineered through

transgenesis, which are also capable of producing enzymes that enhance rate of industrial chemical

reactions.

6.0 ETHICAL CONCERNS

Ethical concerns deals include both intrinsic and extrinsic objections. Intrinsic objections based

heavily on emotions, nevertheless, will not disappear from public belief. Extrinsic objections include

the purpose and consequences of transgenic animal production. The main application of transgenesis

is to develop improve animal welfare outcomes. GMOs have been intentionally designed to suit

human purposes but use of animals in science had always been controversial issue. Some of the

progresses are considered morally unacceptable e.g. cloning. There are some moral or ethical factors

concerned to genetic manipulation based on principle of 3 R’s i.e. Reduction of animal numbers,

refinement of practices and husbandry to minimize pain and distress and Replacement of animals with

non – animal alternatives wherever possible. Ethical concern also focuses in animal welfare concerns,

that is,

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o Use of animals in research causes great suffering to the animals.

o Each biological species has a right to exist as a separate identifiable entity.

o Due to experimentation, it had diminished the sense of identity and individuality.

Xenotransplantation Issues – transplantation or organs from transgenic pigs to humans can spread

Zoonotic disease such as Mad Cow disease, which could have devastating consequences.

7.0 SUMMARY

The emergence of transgenic technology had improved medicine, health, and livestock sector.

Transgenic technology had provided an entire new perception for modulation of organisms at genetic

level. Transgenic animals are created by transfer of foreign gene known as transgene having desired

characteristics. Most common methods for creation of transgenic animals include microinjection,

somatic cell nuclear transfer, embryonic stem cell mediated and retroviral based method.. Transgenic

animals such as sheep, fish and cattle has showed great potentials in medical and agricultural areas.

Transgenic animals such pigs raise ethical concern regarding xenotransplantation.

paper no. : 09 animal cell biotechnology module :10

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